L-glutamate is a excitatory neurotransmitter found in the central nervous system of mammals, and it is known not only to induce rapid neurotransmission between synapses but also to be involved on a higher level in the complex physiological processes of memory and learning. Excitatory neurotransmission between synapses begins with release of glutamate from the presynapse, and fades with rapid glutamate uptake from the synaptic cleft by high-affinity glutamate transporters found in nerve endings and glial cells (Attwell, D. and Nicholls, D., TIPS 68-74, 1991).
Reduced sodium-dependent glutamate uptake activity in portions of patient brains has been reported in several genetic neurodegenerative diseases (Rothstein, J. D. et al., N. Eng. J. Med. 326, 1464-1468, 1992). For this reason, activation and inhibition of glutamate transporter function are becoming objects of focus in connection with such diseases.
In initial steps of research, study of glutamate transporters was carried out primarily using synaptosomes prepared from brain tissue or membrane specimens from the kidney and small intestine. Later, after cloning of sodium-dependent high-affinity glutamate transporter cDNA in 1992, research was conducted from a molecular biological perspective (Pines, G. et al., Nature 360, 464-467, 1992; Storck, T. et al., Proc. Natl. Acad. Sci. USA, 89, 10955-10959, 1992; Kanai, Y. et al., Nature 360, 467-471, 1992). In 1994, the human glutamate transporter gene was cloned and five subtypes, EAAT1 to EAAT5, were categorized (Arriza, J. L. et al., J. Neurosci. 14, 5559-5569; Fairman, W. A. et al., Nature, 375, 599-603, 1995; Arriza, J. L. et al., Proc. Natl. Acad. Sci. USA 94, 4155-4160, 1997).
However, given the low homology of glutamate transporter protein with other neurotransmitter transporters and the difficulty of inferring transmembrane regions based on hydrophobisity, there is still disagreement regarding the 3-dimensional structure and substrate recognition site structure (Grunewald, M. et al., Neuron 21, 623-632, 1998;, Seal, R. P. et al., Neuron 25, 695-706, 2000).
In light of these circumstances, it is desirable to develop various glutamate transporter inhibitors, and especially inhibitors that function as blockers, toward elucidation of the relationship between the glutamate transporter family and neuropathic disorders and neurodegenerative diseases such as epilepsy, Huntington's disease, amyotrophic lateral sclerosis (ALS) and Alzheimer's disease.
As a result of investigation for glutamate uptake inhibitors using synaptosomes according to the prior art, compounds such as threo-β-hydroxyaspartate and CCG-III [(2S,1′S,2′R)-2-(carboxycyclopropyl)glycine], t-2,4-PDC (trans-pyrrolidine-2,4-dicarboxylic acid) and the like have hitherto been identified as glutamate uptake inhibitors, and these are themselves taken up as substrates by transporters and thus act as inhibitors that competitively inhibit glutamate uptake.
The glutamate uptake inhibitors such as kainic acid and dihydrokainic acid were demonstrated by electrophysiological studies to be blockers that inhibit glutamate uptake without themselves being taken up. It was further shown that these compounds act only on EAAT2 (GLT-1 type) of the five EAAT subtypes (Arriza, J. L. et al., J. Neurosci. 14, 5559-5569, 1994). Nevertheless, these compounds have also exhibited strong excitatory effects on ion-channel glutamate receptors.
The present inventors have reported that β-hydroxyaspartate derivatives having substituents at the β-position exhibit an uptake-inhibiting effect for all of the five EAAT subtypes (Lebrun, B. et al., J. Biol. Chem. 272, 20336-20339, 1997; Shimamoto, K. et al., Mol. Pharmacol. 53, 195-201, 1998; Shigeri, Y. et al., J. Neurochem. 79, 297-302, 2001). Among them, it was found that compounds with bulky substituents at the β-position act as blockers for all of the subtypes, inhibiting not only uptake of glutamate but also heteroexchange-based glutamate efflux and sodium ion influx (Chatton, J-Y. et al., Brain Res. 893, 46-52, 2001). In particular, L-threo-β-benzyloxyaspartate (L-TBOA), because of its powerful blocking effect and its lower affinity for glutamate receptor compared to existing inhibitors, has become a standard substance used in glutamate transporter research.